Neurobiology of Disease

Activity-Dependent Tau Protein Translocation to ExcitatorySynapse Is Disrupted by Exposure to Amyloid-BetaOligomers

Marie Lise Frandemiche,1,2 Sandrine De Seranno,1,2 Travis Rush,1,2 Eve Borel,1,2 Aureliane Elie,1,2 Isabelle Arnal,1,2

Fabien Lante,1,2 and Alain Buisson1,2

1INSERM, U836, BP 170, and 2Universite Joseph Fourier, Grenoble Institut des Neurosciences, BP 170, Grenoble Cedex 9, F-38042, France

Tau is a microtubule-associated protein well known for its stabilization of microtubules in axons. Recently, it has emerged that tauparticipates in synaptic function as part of the molecular pathway leading to amyloid-beta (A�)-driven synaptotoxicity in the context ofAlzheimer’s disease. Here, we report the implication of tau in the profound functional synaptic modification associated with synapticplasticity. By exposing murine cultured cortical neurons to a pharmacological synaptic activation, we induced translocation of endoge-nous tau from the dendritic to the postsynaptic compartment. We observed similar tau translocation to the postsynaptic fraction in acutehippocampal slices subjected to long-term potentiation. When we performed live confocal microscopy on cortical neurons transfectedwith human-tau-GFP, we visualized an activity-dependent accumulation of tau in the postsynaptic density. Coprecipitation using phal-loidin revealed that tau interacts with the most predominant cytoskeletal component present, filamentous actin. Finally, when weexposed cortical cultures to 100 nM human synthetic A� oligomers (A�o’s) for 15 min, we induced mislocalization of tau into the spinesunder resting conditions and abrogated subsequent activity-dependent synaptic tau translocation. These changes in synaptic tau dy-namics may rely on a difference between physiological and pathological phosphorylation of tau. Together, these results suggest thatintense synaptic activity drives tau to the postsynaptic density of excitatory synapses and that A�o-driven tau translocation to the spinedeserves further investigation as a key event toward synaptotoxicity in neurodegenerative diseases.

Key words: amyloid-beta oligomers; excitatory synapses; LTP; phosphorylation; synaptic plasticity; tau

IntroductionFibrillar deposits of phosphorylated tau are a characteristic fea-ture of several neurodegenerative diseases, including Alzheimer’sdisease (AD). Increasing evidence suggests that the presence ofneurofibrillary tangles does not cause direct neuronal dysfunc-tion, but rather alterations to physiological tau functions may bekey to disease processes (for review, see Jaworski et al., 2010;Morris et al., 2011). Tau is a highly soluble protein with a pre-dominant expression in neurons (Trojanowski et al., 1989). Alarge proportion of tau is in the axon, where it interacts withmicrotubules (MTs) through its C-terminal microtubule-binding domain to promote MT polymerization and stabiliza-

tion (for review, see Gotz et al., 2013). Accordingly, tau exerts acritical role in the regulation of microtubule-dependent axonaltransport. More recently, numerous studies have revealed novelfunctions for tau, including in neuronal signaling pathways,DNA protection, and synaptic regulation. These studies werebased on the identification of tau interaction with novel molecu-lar partners such as DNA or RNA (Loomis et al., 1990; Sultan etal., 2011), neuronal membranes (Pooler et al., 2012), and synap-tic located proteins such as Fyn or PSD-95 (Ittner et al., 2010;Mondragon-Rodríguez et al., 2012). Subsequently, the associa-tion of tau with synaptic proteins has raised the intriguing possi-bility that tau exerts physiological roles in the synapse and thataltered tau function may be involved in the synaptic dysfunctionthat precedes synaptic loss common across neurodegenerativetauopathies.

Although postsynaptic localization of tau was initiallyidentified under pathological conditions (Hoover et al., 2010),there are several lines of evidence suggesting the involvementof tau in physiological synaptic functions. In 2010, Ittner etal.showed that the presence of tau in the postsynaptic com-partment mediates the targeting of the src family kinase fyn toglutamatergic NMDA receptors. More recently, Chen et al.(2012) suggested that tau participates in spine edification bydemonstrating that reduction of tau expression results in syn-apse loss. Together, these data are consistent with a functional

Received Oct. 3, 2013; revised March 19, 2014; accepted March 25, 2014.Author contributions: M.L.F., S.D.S., T.R., A.E., I.A., F.L., and A.B. designed research; M.L.F., S.D.S., T.R., E.B., A.E.,

I.A., F.L., and A.B. performed research; M.L.F., S.D.S., T.R., E.B., A.E., I.A., F.L., and A.B. contributed unpublishedreagents/analytic tools; M.L.F., S.D.S., T.R., A.E., I.A., F.L., and A.B. analyzed data; M.L.F., S.D.S., T.R., A.E., I.A., F.L.,and A.B. wrote the paper.

This work was supported by the Fondation Neurodis, INSERM, and the Centre National de la Recherche Scienti-fique joint ATIP-Avenir program and the Agence Nationale de la Recherche (ANR MALAAD program). A.E. is sup-ported by a grant from the Region Rhone-Alpes (program CIBLE 2011). We thank N. Sergeant (Lille, France) forproviding us with His-tagged human tau plasmid.

The authors declare no competing financial interests.Correspondence should be addressed to Alain Buisson, Inserm U836, GIN, BP 170, Grenoble Cedex 9, 38042,

France. E-mail: alain.buisson@ujf-grenoble.fr.DOI:10.1523/JNEUROSCI.4261-13.2014

Copyright © 2014 the authors 0270-6474/14/346084-14$15.00/0

6084 • The Journal of Neuroscience, April 23, 2014 • 34(17):6084 – 6097

role for tau in the physiological activation of excitatorysynapses.

Dendritic spines are small protuberances observed on thedendrites that are the physical support of postsynaptic sites ofexcitatory synapses. They are enriched in neurotransmitter re-ceptors, specific scaffolding proteins, and display an abundantactin cytoskeleton responsible for spine morphology (for review,see Hotulainen and Hoogenraad, 2010). Under specific activa-tion patterns, spines undergo profound and long-lasting modifi-cations of morphology and synaptic strength that togetherunderlie synaptic plasticity. Synaptic dysfunction is the best cor-relate of the cognitive decline that characterizes AD, and tau hasbeen implicated in this synaptotoxic effect triggered by amyloid-beta (A�) oligomer exposure. With the emergent idea that taucould penetrate into the synaptic compartment, we investigatedthe mechanisms of tau recruitment to the spines under synapticactivation and A�o exposure. To monitor tau localization, weused subcellular fractionation of both ex vivo (i.e., acute hip-pocampal slices) and in vitro (i.e., cultured cortical neurons) sub-jected to an electrophysiological or chemical long-termpotentiation (LTP). In parallel, we monitored GFP-tagged taucoupled with dynamic fluorescence imaging.

Materials and MethodsPrimary cultures of cortical neurons. Mouse cortical neurons were cul-tured from 14- to 15-d-old OF1 embryos as described previously(Leveille et al., 2008). Cerebral cortices were dissected, dissociated, andcultured in Dulbelcco’s modified Eagle’s medium containing 5% fetalbovine serum, 5% horse serum, and 2 mM glutamine (all from Sigma) on24-well plates (Falcon; Becton Dickinson) for biochemical experiments.Neurons were seeded on 35 mm glass-bottom dishes (MatTek) at a finalconcentration of two cortical hemispheres per dish for confocal experi-ments. All plates, dishes, and coverslips were coated with 0.1 mg/mlpoly-D-lysine and 0.02 mg/ml laminin (Sigma). Cultures were main-tained at 37°C in a humidified atmosphere containing 5% CO2/95% air.After 3– 4 d in vitro (DIV), cytosine arabinoside (AraC, 10 �M; Sigma)was added to inhibit proliferation of non-neuronal cells in cultures usedfor biochemistry experiments; 98% of the cells were considered as neu-ronal. The day before the experiments, cells were washed in DMEM.Treatments were performed on neuronal cultures at 14 –15 DIV.

Neuronal transfection. Transfections were performed on cortical neu-ron cultures after 12 DIV with calcium phosphate precipitation. Growthmedium (DMEM and sera) was removed and kept until the last step oftransfection. Cells were washed for 1–1.5 h in DMKY buffer containingthe following (in mM): 1 kynurenic acid, 0.9 NaOH, 0.5 HEPES, 10MgCl2, plus phenol red 0.05%, pH 7.4. Then, 3.5 �g of the plasmidspEGFP-Tau 2N4R (full-length human nervous system tau) and LifeAct-RFP, a peptide that specifically interacts with filamentous actin (Riedl etal., 2008), were mixed with CaCl2 (120 mM) in HBS containing the fol-lowing (in mM): 25 HEPES, 140 NaCl, and 0.750 Na2HPO4, pH 7.06) andleft for 20 min to precipitate the DNA. Plasmids were then applied to cellsfor 30 min. Transfection medium was replaced with conditioned growthmedium and cultures were returned to the incubator until use at DIV14 –15.

Mutagenesis. Point mutations were performed on pEGFP-Tau 2N4R(full-length human nervous system tau) using the QuikChange kit fromAgilent Technologies.

Fluorescence recovery after photobleaching experiments. Fluorescencerecovery after photobleaching (FRAP) was performed on culturedneurons 48 h after transfection. Images were acquired with an in-verted Nikon Eclipse Ti C2 confocal microscope with a Nikon 60�water objective with a 1.33 numerical aperture. EGFP-tau wasbleached at 405 nm and the fluorescence recovery was measured for80 s (at 1 s/frame). Fluorescent signal analysis was performed with theNikon software Nis.

Time-lapse imaging of transfected neurons. Transfected neurons wereplaced in HBBSS solution containing the following (in mM): 0.110 NaCl,

0.005 KCl, 2 CaCl2, 0.0008 MgSO4, NaH2PO4 1 NaH2PO4, 12 HEPES, 5D-glucose, 25 NaHCO3, and 0.01 glycine (all from Sigma) 1.5–2 h beforeexperiments. Neurons were imaged with a Zeiss LSM 710 confocal im-aging system using a 63� oil-immersion objective and ZEN 2010 soft-ware. Z-stacks were 0.61 �m per step (1 pixel � 0.134 �M) with a 1 AUpinhole at 84 �m. Images were collected immediately before and 15 and30 min after treatment. Evolution of the fluorescence within spine headsin maximum projection was analyzed with MetaMorph software (Mo-lecular Devices). A total of 20 – 40 spines per neurons were followedthroughout treatment for each experiment.

Slice preparation. Hippocampal slices were prepared from 2- to4-month-old male OF1 mice. Mice were cervically dislocated and imme-diately decapitated. Their hippocampi were dissected out and 400-�m-thick transverse slices were cut in ice-cold cutting solution containing thefollowing (in mM): 2.5 KCl, 1.25 NaH2PO4, 10 MgSO4, 0.5 CaCl2, 26NaHCO3, 234 sucrose, and 11 glucose, saturated with 95% O2 and 5%CO2 with a Leica VT1200 blade microtome. After the dissection, sliceswere kept in oxygenated artificial CSF (ACSF) containing the following(in mM): 119 NaCl, 2.5 KCl, 1.25 NaH2PO4, 1.3 MgSO4, 2.5 CaCl2, 26NaHCO3, and 11 glucose 11 at a temperature of 27 � 1°C for at least 1 h.

Electrophysiology recordings. Slices were visualized in a chamber on anupright microscope with transmitted illumination and continuouslyperfused at 1 ml/min with the same oxygenated ACSF at 27 � 1°C.Stimulating electrodes (bipolar microelectrodes) were placed in the stra-tum radiatum to stimulate the Schaffer collaterals pathway. Field EPSPs(fEPSPs) were recorded in the stratum radiatum using a recording glasspipette filled with ACSF and amplified with an Axon Axopatch 200Bamplifier (Molecular Devices). Data were digitized through an AxonDigidata 1440A and acquired with pCLAMP 10 software (MolecularDevices). The initial slope of the fEPSPs was measured to avoid popula-tion spike contamination. For LTP experiments, test stimuli (0.2 mspulse width) were delivered once every 15 s and the stimulus intensitywas set to give baseline fEPSP slopes that were 50% of maximal evokedslopes.

Slices that showed maximal fEPSP sizes �1 mV were rejected. LTP wasinduced by applying 4 trains of 100 stimuli at 100 Hz with an intertraininterval of 5 min. CA1 slices were frozen in liquid nitrogen at the end ofexperiment for subsequent biochemical analyses.

Purification of tau. His-tagged human Tau (1N4R Tau-412 isoform)plasmid was kindly provided by Dr. N. Sergeant (Lille, France). Recom-binant Tau was purified from E. coli BL21 (Invitrogen) on Talon metalaffinity resin (Clontech) according to the manufacturer’s instructions.The protein was further processed by a size exclusion chromatography inBRB80 buffer containing the following (in mM): 80 Pipes, 1 EGTA, and 1MgCl2, pH 6.8, concentrated, and ultracentrifuged at 230,000 � g for 10min. The protein was frozen in liquid nitrogen and stored at �80°C.Protein concentrations were determined by a Bradford assay (Sigma)using BSA as a standard.

In vitro cosedimentation assays. Actin cosedimentation assays wereperformed with purified human platelet actin (Cytoskeleton) resus-pended in buffer G containing the following (in mM): 2 Tris, pH 8.0,0.2 ATP, 0.5 DTT, and 0.1 CaCl2. Then, 5 �M G-actin was polymer-ized for 1 h at room temperature in the absence or in the presence of1 �M Tau in buffer G containing the following (in mM): 50 KCl, 1MgCl2,1 EGTA, and 10 imidazole, pH 7.5. The 20 �l reaction mix-tures were then centrifuged either for 15 min at 100,000 � g or for 10min at 15,000 � g. At low-speed centrifugation, filamentous actinremains in the supernatant and only filamentous actin (F-actin) bun-dles are pelleted, whereas the high-speed centrifugation precipitatesall F-actin forms. Supernatants and pellets fractions were recoveredand subjected to 10% SDS-PAGE gel, followed by Coomassie bluestaining.

Subcellular fractionation. Cultured neurons or hippocampal CA1 sliceswere homogenized in cold buffer containing 0.32 M sucrose and 10 mM

HEPES, pH 7.4. Samples were maintained at 4°C during all steps of theexperiment. Homogenates were cleared at 1000 � g for 10 min to removenuclei and large debris. The resulting supernatants were concentrated at12,000 � g for 20 min to obtain a crude membrane fraction, which wasthen resuspended twice (4 mM HEPES, 1 mM EDTA, pH 7.4, 20 min at

Frandemiche et al. • Activity-Dependent Tau Protein Translocation J. Neurosci., April 23, 2014 • 34(17):6084 – 6097 • 6085

12,000 � g). Then, the pellet was incubated (20 mM HEPES, 100 mM

NaCl, 0.5% Triton X-100, pH 7.2) for 1 h at 4°C with mild agitation andcentrifuged at 12,000 � g for 20 min to pellet the synaptosomal mem-brane fraction. The supernatant was collected as the non-postsynapticdensity membrane fraction (non-PSD) or Triton-soluble fraction. Thepellet was then solubilized (20 mM HEPES, 0.15 mM NaCl, 1% TritonX-100, 1% deoxycholic acid, 1% SDS, pH 7.5) for 1 h at 4°C and centri-fuged at 10,000 � g for 15 min. The supernatant contained the PSD orTriton-insoluble fraction. The integrity of non-PSD was verified by im-munoblotting for synaptophysin, which was enriched in the non-PSDfraction, and the PSD fraction was confirmed by the immunoblotting ofPSD-95 enriched in this compartment.

A� oligomerization and fibrillogenesis. Recombinant A�1– 42 peptide(Bachem) was resuspended in 1,1,1,3,3,3-hexafluoro-2-propanol(HFIP) to 1 mM until complete resuspension as described previously(Stine et al., 2003). A�o’s were prepared by diluting A� to 1 mM in DMSOand then to 100 �M in ice-cold HEPES and bicarbonate-buffered salinesolution (HBBSS) with immediate vortexing and bath sonication, andthen incubated at 4°C for 24 h with mild agitation.

Synaptic activation and actin cytoskeleton disruption. Cultures wereplaced in DMEM 24 h before the experiment. Synaptic activation wasinduced at 37°C with 50 �M bicuculline (Bic), a GABAa receptor antag-onist, and 2.5 mM 4-amino-pyridine, a weak potassium channel blocker(all from Tocris Bioscience) for 15 min (Leveille et al., 2008). Actinstabilization was induced by jasplakinolide (Santa Cruz Biotechnology)application at 1 �M (Lazaro-Dieguez et al., 2008).

Immunoprecipitation. After treatment, primary neurons were rapidlytransferred on ice to remove the media and immediately extracted withfresh buffer (100 mM Na2HPO4, 100 mM NaH2PO4, pH 7.2, 2 mM ATP, 2mM MgCl2, phosphatase, and protease inhibitor mixture; Sigma). Equalamounts of protein were incubated with biotin-XX-phalloidin (Molec-ular Probes) 0.45 unit/condition with gentle rocking for 1 h at roomtemperature. Thereafter, Dynabeads M-280 Streptavidin (Invitrogen)were added to the complex formed by filamentous actin-biotin-XX phal-loidin and incubated for 30 min at room temperature using gentle rota-tion. The actin-coated beads were separated with a magnet for 2–3 min,washed several times in the buffer, and boiled in 1� loading buffer con-taining 124 mM Tris, 2% SDS, 10% glycerol, 1% �-mercaptoethanol, and1% bromophenol blue for 5 min. Samples were stored at �20°C untilused.

Immunoblotting. Samples in loading buffer were boiled for 5 min andequal amounts of proteins (5–10 �g) were resolved on a 4 –12% or4 –20% gradient Bis-Tris polyacrylamide precast gels (Bio-Rad) in dena-turing conditions. Proteins were transferred to a polyvinylidene difluo-ride membrane (Millipore) for 2 h at 4°C. Membranes were blocked withTris-buffered saline (10 mM Tris, 150 mM NaCl, pH 7.4) containing0.01% Tween 20 and 5% nonfat dry milk for 1 h at room temperature.Membranes were then incubated overnight at 4°C with the primary an-tibodies tau (1:50,000 dilution, A0024; Dako); tau phospho serine 404and tau phospho tyrosine 205 (1:1000 dilution, PA1–14422, PA1–14426,PA1–14415; Thermo Scientific); actin (1:2000, A2066, Sigma); GluA1,GluN2B, GluN2A, and synaptophysin (1:1000 dilution, A1504,MAB5778, 04 –901, MAB368; Millipore); Fyn (1:500 dilution, 15-sc-434;Santa Cruz Biotechnology); and PSD-95 (1:1000 dilution, clone 6G6 –1C9 ab2723; Abcam). Membranes were incubated with the HRP-conjugated secondary antibodies rabbit (1:40,000) and mouse (1:5000)(both from Sigma) for 45 min at room temperature. Specific proteinswere visualized with an enhanced chemiluminescence ECL DetectionSystem (Bio-Rad). Chemiluminescence detections were performed withthe Bio-Rad Chemidoc system and analyzed with the software Imagelab(Bio-Rad).

Statistics. Results are expressed as the mean � SEM from independentbiological samples. Statistical analyses were performed with GraphPadPrism 6.0 software by two-way ANOVA followed by Bonferroni’s test forconfocal analysis, one-way ANOVA for phalloidin precipitation analysis,and Student’s t test for Western blot analysis.

ResultsAxonal and dendritic localization of tau in primary corticalneurons under resting conditionsTo characterize the localization of tau in cultured neuronsunder resting conditions (14 DIV), we transfected full-lengthtau 2N4R-EGFP and LifeAct-RFP, the latter of which encodesa small, 17 aa peptide that binds specifically to filamentousactin (Riedl et al., 2008). In neurons, filamentous actin isknown to be enriched to the synaptic compartment (Star et al.,2002). By using LifeAct-RFP instead of actin-GFP, we main-tained actin stoichiometry. We first verified the localization ofLifeAct-RFP in the synaptic compartment by cotransfecting asynaptic scaffold protein, PSD-95-YFP. The merged imageshowed that filamentous actin and PSD-95 are both localizedin dendritic spines (Fig. 1A). We then cotransfected LifeAct-RFP with EGFP-tau in cortical neurons and observed thatEGFP-tau fluorescence is restricted to axonal and dendriticshafts in the resting condition (Fig. 1B). Only a minimal num-ber of spines displayed traces of EGFP-tau (Fig. 1B).

Synaptic activation induces translocation of tau to thesynaptic compartmentTo determine how and under which circumstances tau entersinto the postsynaptic compartment, we monitored EGFP-tau flu-orescence under various conditions. First, we induced a long-lasting synaptic activation by exposing cultured cortical neuronsto Bic (50 �M), a GABAergic antagonist, in the presence of4-aminopyridine (4-AP, 2.5 mM), a weak potassium channelblocker that prevents neuronal repolarization (Leveille et al.,2008), for 30 min (Fig. 2A). In control conditions, EGFP-taushowed a weak but detectable fluorescence in �60% of the spine,but the calculation mode used to quantify the imaging data (�F/F0) brings our quantification to zero to highlight changes frombase line fluorescence. As early as 15 min after the beginning ofthe treatment, we measured an increase in EGFP-tau fluores-cence in dendritic spines from 0 � 0.04 to 0.52 � 0.06, whichstabilized after 30 min to 0.64 � 0.05 (***p � 0.0001, 2-wayANOVA; Bic/4-AP: n � 5, N � 162 spines; control n � 3, N � 40spines; Fig. 2B) and colocalized with LifeAct-RFP fluorescence.In contrast, the evolution of EGFP-tau fluorescence during a 30min vehicle application showed no detectable change (Fig. 2B).Recognizing that plasmid-driven expression of EGFP-tau maymodify tau cellular distribution, we studied the localization ofendogenous tau in neurons subjected to synaptic activation. Forthis study, we use a primary cortical cultures system at a densitythat permits biochemical analysis but also renders immunohis-tochemistry impractical because it is very difficult to distinguishindividual immunostained spines. Western blot analysis of taufrom total neuronal lysate showed no change after 15 and 30 minBic/4-AP application (Fig. 2C). Then, we isolated the non-PSDfraction (i.e., Triton-soluble fraction) from the PSD-enrichedfraction (i.e., Triton-insoluble fraction) and audited the presenceof synaptophysin and PSD-95 in each to verify the fractionationprotocol. As expected, synaptophysin appeared only in the non-PSD fraction and PSD-95 only in the PSD-enriched fraction.Next, we performed Western blot analysis of several synapticproteins to assess the effect of our stimulation protocol (Fig. 2D).No change in tau expression was detected in the non-PSD frac-tion. In contrast, tau expression in the PSD fraction showed asmall amount in resting conditions, which increased 1.5-fold af-ter 15 min of synaptic activation (***p � 0.0001, 2-tailed Stu-dent’s t test; control 20.62 � 1.235 vs Bic/4-AP 30.92 � 1.799,N � 13 independent cultures; Fig. 2E). On the same PSD-positive

6086 • J. Neurosci., April 23, 2014 • 34(17):6084 – 6097 Frandemiche et al. • Activity-Dependent Tau Protein Translocation

fractions, we analyzed the variation of synaptic markers thathave been identified as potential tau-interacting partners (Leeet al., 1998; Ittner et al., 2010; Mondragon-Rodríguez et al.,2012). After synaptic activation, we detected increases in PSD-95(*p � 0.0249, 2-tailed Student’s t test; control 19.10 � 2.557 vsBic/4-AP 26.9 � 1.894, N � 9; Fig. 2E) and the src family tyrosinekinase Fyn (**p � 0.0023, 2-tailed Student’s t test; control19.42 � 1.337 vs Bic/4-AP 26.54 � 1.130, N � 6; Fig. 2E). TheAMPA receptor subunit GluA1, which is recruited to the mem-brane during synaptic plasticity (Malinow and Malenka, 2002),was also increased in the PSD fraction (**p � 0.0026, 2-tailedStudent’s t test; control 1884 � 1930 vs Bic/4-AP 2863 � 1.95,N � 9; Fig. 2E). The actin cytoskeleton is highly reorganizedduring synaptic activation, as described previously (Star et al.,2002). Here, we saw an increase in actin content restricted to thePSD fraction (*p � 0.0101, 2-tailed Student’s t test; control22.18 � 1.913 vs Bic/4-AP 29.62 � 1.517, N � 7 independentcultures; Fig. 2E). Therefore, during a long-lasting synaptic acti-vation, we observed an increase in tau, fyn, actin, GluA1, andPSD-95 content in the PSD-positive fraction, which is consistentwith the characteristic features of synaptic plasticity (Ehlers,2003). These results suggest that tau translocates from the den-

dritic shaft to the synapse during activa-tion and probably takes part in theactivity-driven synaptic reorganizationthat underlies synaptic plasticity. To gen-eralize these findings, we chose to confirmthese results in an ex vivo model of synap-tic plasticity: LTP in hippocampal slices(Fig. 3A). We performed synaptosomalfractionation of microdissected CA1 re-gions taken from potentiated hippocam-pal slices. We observed a similar LTP-induced increase in tau content within thePSD-enriched fraction from CA1 synap-tosomes (29.86 �4.86 to 70.15 � 4.86,**p � 0.0011; Fig. 3B). As expected, actinand GluA1 were also increased, strength-ening the idea that tau is involved in syn-aptic reorganization processes necessaryfor synaptic plasticity (34.31 � 2.518 to64.92 �2.518, ***p � 0.0002; 34.31 �8.15 to 65.70�8.15, *p � 0.0346; Fig. 3C).

Tau interacts with filamentous actin inthe synapseAlthough it was initially described as amicrotubule-associated protein (MAP),tau has been shown to interact with othermolecular partners in the PSD fraction,such as fyn or PSD-95 (Ittner et al., 2010;Mondragon-Rodríguez et al., 2012).Among several synaptically located pro-teins, actin is of particular interest for itspotential capacity to interact with humantau (Fulga et al., 2006; Yu and Rasenick,2006; He et al., 2009). We investigatedwhether tau interacts with actin, the mostpredominant cytoskeleton element of thesynapse. Purified G-actin was polymer-ized with the 1N4R tau isoform and cen-trifuged at 100,000 � g (Fig. 4A) or15,000 � g (Fig. 4B). Although tau alone

were observed only in the supernatant in both experimental con-ditions, ruling out a nonspecific coaggregation, we found that taucoprecipitated with the pellet obtained from high- and low-speedcentrifugation, illustrating its direct interaction with both F-actin(Fig. 4A) and actin bundles (Fig. 4B). It is worth noting that thefilamentous nature of actin in the presence of tau was confirmedby electron cryomicroscopy (data not shown). We also per-formed transfection with 1N4R-GFP and we observed similarsynaptic-activity-dependent translocation as observed with thefull-length tau (Fig 4C). Similarly, a phalloidin precipitation ofneuronal lysate that selectively collected the neuronal F-actin andits binding partners revealed the presence of tau by Western blot-ting. This result illustrates that tau interacts with F-actin in neu-rons (Fig. 5A). After synaptic activation, we observed an increasein the amount of F-actin harvested (***p � 0.0003, 2-tailed Stu-dent’s t test; control 17.67 � 2.08; Bic/4-AP 31.76 � 1.882, N � 7independent cultures; Fig. 5B) and a concomitant increase in tau(***p � 0.0002, control 16.91 � 1.843; Bic/4-AP 31.49 � 2.028,N � 7 independent cultures). These results show that the amountof tau collected is proportional to neuronal F-actin content, sug-gesting a close link between F-actin and tau. Next, we studied theimpact of pharmacological manipulations of actin organization

Figure 1. LifeAct colocalizes with PSD-95-YFP in dendritic spines and displays a limited colocalization with EGFP-Tau. A,Confocal imaging of dendritic spines of primary murine cortical neuronal cultures (14 DIV) cotransfected with LifeAct-RFP andPSD-95-YFP 48 h before imaging. Merge images show the colocalization (yellow, white arrows) of PSD-95 and filamentous actin indendritic spines. B, Dendritic spines of cortical neuron culture cotransfected with LifeAct-RFP and EGFP-Tau. Merged images showonly limited colocalization of EGFP-Tau in the indicated dendritic spines (white arrows). Scale bars: top, 10 �m; bottom, 5 �m.

Frandemiche et al. • Activity-Dependent Tau Protein Translocation J. Neurosci., April 23, 2014 • 34(17):6084 – 6097 • 6087

on synaptic EGFP-tau fluorescence localization. For this, wetreated our primary cortical neurons with jasplakinolide (1 �M),a compound that promotes actin polymerization (Lazaro-Dieguez et al., 2008), or with a latrunculin A (at 500 nM), a com-pound that depolymerizes F-actin into soluble globular actin (G-actin; Coue et al., 1987; Fig. 5C). After jasplakinolide application,we observed a large increase in synaptic EGFP-tau fluorescence(***p � 0.0001, 2-way ANOVA; �F/F0, 0.64�0.05 after 15 min,0.59�0.06 after 30 min; control n �40, N � 3; jasplakinolide, n �106, N � 4; Fig. 4D). Latrunculin treatment produced rapid actindepolymerization and the corresponding disappearance of

LifeAct-RFP fluorescence in every spine studied; no synapticEGFP-tau fluorescence was observed (data not shown).

We analyzed actin and tau in the PSD-enriched fraction fromprimary cortical neurons treated with jasplakinolide (Fig. 5E).We observed that increased neuronal F-actin content promotesconcurrent tau enrichment (*p � 0.0150, 2-tailed Student’s t test;control 17.49 � 0.7755 vs jasplakinolide 27.02 � 2719, N � 4independent cultures; Fig. 5F). GLUA1, the membrane traffick-ing of which is known to be actin dependent, was increased (*p �0.0279, 2-tailed Student’s t test; control 16.91 � 1015 vs jas-plakinolide 31.00 � 4.778, N � 4 independent cultures). The

Figure 2. Synaptic activation induced by Bic/4-AP triggers translocation of tau into dendritic spines of primary cortical neuron cultures. A, Confocal imaging of cortical neurons (14 DIV)cotransfected with EGFP-Tau and LifeAct-RFP. Left, Spatial repartition of EGF-Tau (green) and LifeAct-RFP (red) in nonactivated neuron. The merged image displays only minor colocalization(yellow). Scale bar, 5 �m. Right, Higher magnification of EGFP-Tau, LifeAct in dendritic spines (designated box in the left) of a neuron after synaptic activation triggered by 15 and 30 min of Bic/4APexposure. The merged image shows the activity driven colocalization of tau/LifeAct (yellow) in the activated dendritic spine after 15 and 30 min Bic/4-AP exposure. Scale bar, 1 �m. B, Quantificationof fluorescence intensity mediated by EGFP-Tau in the head of spines during synaptic activation (Bic/4-AP treatment). The graph represents the evolution of �F/F0 generated by EGFP-Tau in neuronssubjected to synaptic activation or not. Statistical analysis was performed by two-way ANOVA followed by Bonferroni’s posttest (mean � SEM, ***p � 0.0001, control n � 40 spines on 3independent cultures; Bic/4-AP n � 162 spines on 5 independent cultures). C, Representative Western blot of endogenous tau expression in a whole lysate extract of cortical neurons (14 DIV)exposed to Bic/4-AP for 15 and 30 min. Quantification of tau expression normalized to actin levels does not display any change. D, Representative Western blots of protein expression from primarycortical cultures after fractionation. Presynaptic protein (synaptophysin), postsynaptic proteins (PSD-95, GluA1, GluN2A, GluN2B, Fyn), cytoskeletal protein (actin), and tau in non-PSD fraction(Triton-soluble fraction) and PSD-fraction (Triton-insoluble fraction) on primary cortical neurons treated 15 min with Bic/4-AP. E, Quantitative analysis of PSD-95, GluA1, GluN2B, GluA1, Fyn, actin,and tau in the PSD fraction under control and Bic/4-AP (15 min) treated conditions is shown in graphs (mean � SEM, 2-tailed Student’s t test,***p � 0.001, **p � 0.0026, *p � 0.0101, N � 7independent cultures).

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amount of Fyn in the PSD was decreased (*p � 0.0265, 2-tailedStudent’s t test; control 27.25 �5.003 vs jasplakinolide 11.71 �1.786, N � 4 independent cultures). Together, these results sug-gest that tau translocation to the synapse depends on the F-actinstabilization that promotes their interaction.

A� oligomers disrupt the link between synaptic activationand tau recruitment to the synapseIn the context of AD, recent studies have shown that synaptotox-icity driven by A�o exposure relies on the presence of tau (Rapo-port et al., 2002; Roberson et al., 2007; Ittner et al., 2010). Tostudy this pathological regulation of tau, we exposed EGFP-tau-and LifeAct-RFP-expressing cultures to 100 nM synthetic humanA�o’s (1– 42; Fig. 6A). We observed a delocalization of tau in thesynapse within 15 min after A�o treatment (***p � 0.001; con-trol n � 40, N � 3, A�o 100 nM for 15 min 0.52� 0.04, for 30 min0.71 � 0.05, n � 79, N � 5 independent cultures; Fig. 6B). Weconfirmed these results with endogenous tau by Western blotanalysis of PSD-enriched fractions (Fig. 6C). A�o exposure in-duced a translocation of tau into the PSD fraction (***p �0.0002, 2-tailed Student’s t test; control 20.12 � 1228 vs A�o29.74 � 1.748, N � 12 independent culture). There was also anincrease of PSD-95 (***p � 0.0006, 2-tailed Student’s t test; con-trol 19.10 � 2.557 vs A�o 33.3 � 2153, N � 9 independentculture), GluA1 (**p � 0.0078, 2-tailed Student’s t test; control18.84 � 1.930 vs A�o 26.22 � 1.475, N � 9 independent culture)

and fyn (**p � 0.0041, 2-tailed Student’s t test; control 19.42 �1.337 vs A�o 29.67 � 2.181, N � 6 independent cultures; Fig.6D). Then, we evaluated the effect of a synaptic activation afterthe A�o treatment (Fig. 7A). Preceded by 15 min A�o treatment,synaptic activation disrupted LifeAct-RFP fluorescence, suggest-ing an alteration of F-actin organization and no additional EGFP-tau recruitment at the synapse. We confirmed these results onendogenous proteins by Western blot analysis on PSD-positivefractions (Fig. 7B). After A�o treatment, synaptic activation didnot trigger any increase in synaptic markers and in fact decreasedsynaptic actin (***p � 0.0009, 2-tailed Student’s t test; A�o29.64 � 1.495, A�o Bic/4-AP 18.56 �2.030, N � 7 indepen-dent cultures; Fig. 7C), PSD-95 (***p � 0.0007, 2-tailed Student’st test; A�o 33.37 � 2.153, A�o Bic/4-AP 19.25 �2.550, N � 7independent cultures) and tau levels (**p � 0.0014, 2-tailed Stu-dent’s t test; A�o 29.74� 1.748, A�o Bic/4-AP 20.68 �1.751,N � 12 independent cultures). If the spines’ protein content didnot display any significant difference, these treatments are asso-ciated with a profound modification of the spine structure, asrevealed by the morphological analysis performed on corticalculture transfected with a plasma membrane marker tagged witha GFP (Fig. 7D). Finally, we performed a phalloidin precipitationassay after a 15 min A�o treatment on our neuron culture (Fig.8A) and observed that tau/F-actin content was increased (**,*p �0.05 relative to control, #p � 0.05 relative to A�o, 1-wayANOVA; control 15.45 � 1.529, A�o 32.90 � 3.181, A�o Bic/

Figure 3. LTP in hippocampal slices triggers translocalization of tau to CA1 dendritic spines. A, Representative curve of fEPSP slope in percentage of baseline illustrating the LTP induction in CA1region of hippocampus slices. Sample EPSPs are shown before (1) and after (2) LTP induction. B, Representative Western blots of synaptic protein expression from the CA1 region of hippocampalslices subjected to LTP protocol. C, Quantifications of Western blot from GluA1, actin, and tau in control and LTP conditions (mean � SEM, 2-tailed Student’s t test, *p � 0.0346, **p �0.0011,***p � 0.0002, N � 4 independent slices).

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4-AP 20.18 � 2671 for actin; control 16.34 � 2.618, A�o 31.77 �1.952, A�o Bic/4-AP 17.70 � 4.080 for tau, N � 5 independentcultures Fig. 8B). A subsequent synaptic activation did alter tauinteraction with F-actin.

Pool of synaptic tau evoked by A�o is more dynamic and hasa distinct phosphorylation state from that induced bysynaptic activationBecause image acquisition for live imaging of EGFP-tau was per-formed at a relatively slow rate to accumulate fluorescence inten-sity, we did not have any direct measurement of tau turnover inthe spines. To further understand the mechanisms by which A�oexposure and the synaptic activation induced translocation of tauto the synapse, we investigated tau dynamics using a FRAP tech-nique. On neurons transfected with EGFP-tau and LifeAct-RFP, we induced tau translocation to spine by synapticactivation, and then photobleached the GFP signal of individ-ual spine heads (Fig. 9A) and measured tau fluorescence,which resulted in a recovery half-life of 4.829 s and a plateau of50.83% (Fig. 9B). The recovery curves indicated 3 pools of tau:a mobile, unbleached fraction comprising 9.72 � 1.03%, adynamic fraction at 47.08 � 3.06%, and a stable, unrecover-

able fraction at 42.75 � 2.78%. This stable fraction suggeststhat a large portion of tau is anchored in the spine. When weinvestigated A�o-driven tau translocation to the synapse, wedid not see any change in half-life recovery (4.729 s) fromthose measured with synaptic activation. However, the plateauvalue was drastically modified (71.20%), illustrating that,whereas A�o induced tau translocation and subsequently itsinteraction with actin filament, the resulting synaptic tau is less sta-ble. Comparison between synaptic activation and A�o showed asignificant difference between their dynamic fractions (***p �0.0001, 2-tailed Student-t test; Bic/4AP dynamic fraction 47.09�3.063, n � 21 spines on 4 independent cultures; A�o 68.49 � 2.64,n � 24 on 4 independent cultures; Fig. 9C).

Tau is a phosphorylated protein containing 80 potentialphosphorylation sites (Sergeant et al., 2005). Alterations of taulocalization in disease states are associated with abnormalphosphorylation of tau (Hoover et al., 2010; Tai et al., 2012;Merino-Serrais et al., 2013); therefore, we investigated thephosphorylation state of tau after synaptic activation or A�oexposure (Fig. 9D). We chose to investigate the phosphoryla-tion states of two representatives residues that have both beendescribed as sites of physiological and pathological phosphor-

Figure 4. Tau interacts with actin in purified system. Cosedimentation of actin filaments with tau. Actin was polymerized at 5 �M for 1 h at room temperature in the absence or in the presenceof 1 �M tau. The reaction mixtures were then centrifuged at 100,000 � g to sediment actin filaments (A) or at 15,000 � g to precipitate only actin bundles (B). Supernatants (SN) and pellets (P)were applied to 10% SDS-PAGE gel and proteins were stained with Coomassie blue. C, Confocal imaging of cortical neurons (14 DIV) cotransfected with EGFP-Tau1N4R and LifeAct-RFP. Left, Spatialrepartition of EGFP-Tau1N4R (green) and LifeAct-RFP (red) in untreated neurons. Scale bar, 5 �m. Right, Higher magnification of EGFP-Tau1N4R and LifeAct-RFP in a dendritic spine neuron afterBic/4-AP treatment (15 and 30 min, designated box in the left) by 15 and 30 min of Bic/4-AP treatment. The merged image shows colocalization of F-actin and tau. Scale bar, 1 �m. D, Quantificationof fluorescence intensity mediated by EGFP-Tau1N4R and Tau2N4R in the head of spines during synaptic activation (Bic/4-AP treatment). The graph represents the evolution of �F/F0 generated byEGFP-Tau2N4R and EGFP-Tau1N4R in neurons subjected to synaptic activation or not. Statistical analysis was performed by two-way ANOVA followed by Bonferroni’s posttest (mean�SEM, ***p�0.0001 vs control, ###p � 0.0001 vs Tau-2N4R; control n � 40 spines on 3 independent cultures; Bic/4-AP Tau2N4R n � 162 spines on 5 independent cultures, Bic/4AP Tau 1N4R n � 54 spines on2 independent cultures).

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ylation: one located in the proline rich domain, Thr-205, andone located in the C terminus domain, Ser-404 (Sergeant et al.,2005; Merino-Serrais et al., 2013). Thr-205 phosphorylated-tau was only increased under synaptic activation in the PSDfraction (control 24.57 � 0.9754 vs Bic/4-AP 38.90 � 1.936;Fig. 9E), whereas it was decreased after A�o treatment (con-trol 24.57 � 0.9754 vs A� 13.64 � 2.416). Synaptic activationafter A�o exposure did not produce any significant Thr-205phosphorylation of tau (A� Bic/4-AP 22.89�2.796 vs Bic/4-AP 38.90 � 1.93 vs A� 13.64�2.50). Conversely, only A�oexposure promoted significant tau phosphorylation on Ser404 (**p � 0.05, 1-way ANOVA; control 15.67 � 2.418 vs A�o32.65 � 3.76 vs Bic/4-AP 26.75 � 1.17 vs A�o Bic/4-AP24.97 � 4.48, N � 4). These results revealed that, althoughsynaptic activation or A�o promote tau translocation to PSD

fractions, the synaptic tau displays a different phosphorylationprofile that may be responsible for the conditional tau prop-erties observed. Finally, to investigate whether the differencein tau retention in the spine was related to differential phos-phorylation, we overexpressed nonphosphorylatable tau mu-tant EGFP-Tau T205A or EGFP-Tau S404A in neuronsexposed to synaptic activation or to A�o exposure. We ob-served that synaptic activation promoted EGFP-Tau T205Atranslocation to the spine but FRAP experiments revealed ashorter tau turnover time in the spine (Fig. 9B), whereas A�o-driven translocation to the spine was no longer observable inEGFP-Tau S404A-transfected neurons (Fig. 9 F, G). These ex-periments highlight the pivotal role of these phosphorylationsin tau translocation features to the spine.

Figure 5. Tau interacts with F-actin in the synaptic compartment. A, Representative Western blot showing F-actin-associated tau in cultured cortical neurons treated for 15 min with Bic/4-AP.Cell lysate was collected after treatment and F-actin precipitated with phalloidin as described in the Materials and Methods. B, Quantifications of actin and tau in control and Bic/4-AP condition(mean � SEM, 2-tailed Student’s t test, ***p � 0.001, N � 7 independent cultures). C, Confocal imaging of cortical neurons (14 DIV) cotransfected with EGFP-Tau and LifeAct-RFP treated withjasplakinolide (1 �M), a specific agent that stabilizes F-actin. Left, Cropped view of dendritic branch of transfected neuron under control condition. Scale bar, 5 �m. Right, Higher magnification ofEGFP-Tau and LifeAct-RFP in dendritic spines (cropped view of dotted box in the left) of neuron before and 15 and 30 min after jasplakinolide (1 �M) treatment. The merged image showscolocalization of EGFP-Tau and LifeAct-RFP (white arrows). Scale bar, 1 �m. D, Quantification of the fluorescence intensity variation (�F/F0) of EGFP-Tau in the head of spines during actin networkstabilization (jasplakinolide treatment). Statistical analysis was performed by two-way ANOVA followed by Bonferroni’s posttest (mean � SEM, ***p � 0.0001, control n � 40, N � 3 independentcultures, jasplakinolide n � 106, N � 4 independent cultures). E, Representative Western blots of protein expression from PSD-enriched fraction primary cortical cultures treated or not withjasplakinolide (1 �M, synaptophysin [SYN], PSD-95, GluA1, Fyn actin, and tau in PSD-fraction (Triton-insoluble fraction)). F, Quantitative analysis of Western blots of PSD-95, GluA1, actin, and tauin PSD fraction (mean � SEM, 2-tailed Student’s t test, **p � 0.01, *p � 0.05, N � 4 independent cultures).

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DiscussionAlthough tau has been extensively studied for its role as an axonalMAP, the discovery of its synaptic localization under pathologicalconditions has raised extensive questions on the consequence ofthis mislocation (Ittner and Gotz, 2011). Here, we report that taudisplays a widespread distribution in the dendrites and respondsto synaptic input by trafficking from the dendritic shaft to thepostsynaptic compartments. The trafficking of tau occurs underpharmacological and electrophysiological synaptic plasticitystimulation paradigms in primary culture and acute hippocam-pal slices, respectively. This activity-dependent tau translocationis concomitant with major recompositions of the postsynapticprotein content that results in an increase in PSD-95, glutama-tergic AMPA receptors, GluA1, actin, and tau’s proposed partner,Fyn kinase. Together, our results are consistent with tau having a

postsynaptic role in the organization that sustains synaptic plas-ticity. Further, we demonstrate that, once translocated to thesynapse, tau interacts with the most predominant cytoskeletonelement of the postsynaptic structure: F-actin. This interaction isconsistent with a possible implication in the complex cascadeleading to synaptic potentiation. Further supporting this hypoth-esis, we observed that pharmacological stabilization of F-actinwith jasplakinolide also induces synaptic tau trafficking, whichsuggests a link between F-actin and tau. Interestingly, we foundthat by exposing cortical neurons to synthetic A�o, tau translo-cates to spines without induced synaptic activation. This mislo-cation of tau into the spine heads is a characteristic featureobserved in AD (Tai et al., 2012) and frontotemporal dementia(FTD) and parkinsonism linked to chromosome 17 (FTDP-17)and tau P301L mutation (Hoover et al., 2010). Synaptic activa-

Figure 6. A�o treatment in primary cortical neuron cultures induces synaptic mislocalization of EGFP-Tau. A, Confocal imaging of cortical neurons (14 DIV) cotransfected with EGFP-Tau(full-length human tau) and LifeAct-RFP. Left, Spatial repartition of EGFP-Tau (green) and LifeAct-RFP (red) in nontreated neuron. Scale bar, 5 �m. Right, Higher magnification of EGFP-Tau andLifeAct-RFP in dendritic spine neuron after A� treatment (15 and 30 min, designated box in the left) by 15 and 30 min of A�o 100 nM treatment. The merged image shows colocalization of F-actinand tau (yellow, area indicated with white arrows). Scale bar, 1 �m. B, Quantification of the fluorescence intensity of EGFP-Tau in the head of spines during the A�o treatment. The graph representsthe evolution of �F/F0 generated by EGFP-Tau induced by A�o 100 nM treatment. Statistical analysis was performed by two-way ANOVA followed by Bonferroni’s posttest (mean � SEM, ***p �0.001, control n � 40, N � 3 independent cultures; A�o 100 nM n � 79, N � 5 independent cultures). C, Representative Western blots of protein expression from fractionated primary corticalcultures treated or not with A�o (100 nM) for 30 min. Presynaptic protein (synaptophysin), postsynaptic proteins (PSD-95, GluA1, GluN2A, GluN2B, Fyn), cytoskeletal protein (actin), and tau inPSD-fraction (Triton-insoluble fraction) were analyzed. D, Quantitative analysis of PSD-95, GluA1, GluN2B, GluA1, Fyn, actin, and tau in PSD fractions (mean � SEM, 2-tailed Student’s t test, ***p �0.001, **p � 0.01, n � 7 independent cultures). E, Percentage of positives and negatives spines in EGFP-Tau-transfected neurons before and during synaptic activation or A�o treatment (Bic/4-APN � 3, n � 130 spines; A�o N � 3, n � 142; control N � 6, n � 272; ***p � 0.0001, **p � 0.01).

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tion subsequent to A�o did not further increase tau content intothe dendritic spines, which suggests that A�o exposure disruptsthe activity-dependent enrichment of tau concomitant with thepreclusion of synaptic plasticity.

The identification of activity-driven translocation of tau to thesynaptic compartment is consistent with Ittner et al. (2010), whodescribed tau function as carrying the Src kinase Fyn to the syn-aptic compartment after synaptic activation. The present find-ings also add to the growing body of evidence consistent with awide distribution of neuronal tau in the dendrites (Tashiro et al.,1997) and the axon (Mandell and Banker, 1996). Tau is a wellknown MAP and microtubules have been shown to penetrateinto spines (Jaworski et al., 2009; Merriam et al., 2013), whichstrongly suggests that microtubules are involved in the observedtranslocation. Different mechanisms may account for the distri-bution of tau in dendritic spine, for example, cotransport withmicrotubule fragments or by motor transport or diffusion. The

simplest, and therefore most likely, scenario is one in which tauenters spines with microtubules. After being liberated from MTs,tau may bind to actin filaments, thereby anchoring tau within thepostsynaptic compartment. This hypothesis is supported by pre-vious work from Ittner et al. (2010), which showed that removingthe MT-associated domain from tau prevents the synaptic local-ization of tau and its binding partner Fyn. When tau is truncatedto contain only the N-terminal and proline-rich domains, tau isunable to localize to dendrites. Because this truncated form of tauis still able to bind Fyn, the exclusion of tau from spines alsoprecludes fyn localization to the postsynaptic compartment. Asan active transporter of synaptic proteins (e.g., AMPARs; Kneus-sel and Wagner, 2013), myosin may also carry tau to the spinesalong a cytoskeletal track. Currently, only one study suggests aninteraction between tau and myosin based on their colocalizationwithin neurofibrillary tangles found in AD and FTD patients’brains (Feuillette et al., 2010). Moreover, myosin VI was shown to

Figure 7. A�o treatment on primary cortical neuron cultures induces synaptic mislocalization of endogenous tau that is disrupted by synaptic activation. A, Confocal imaging of cortical neurons(14 DIV) cotransfected with EGFP-Tau and LifeAct-RFP. Left, Spatial repartition of EGFP-Tau (in green) and LifeAct-RFP (in red) in untreated neurons. Scale bar, 5 �m. Right, Higher magnificationof EGFP-Tau and LifeAct-RFP in a dendritic spine (designated box in the left) neuron showing the effect of 15 min of A� on tau localization (green) and actin cytoskeleton (red) followed by 15 minof Bic/4-AP. Scale bar, 1 �m. B, Representative Western blots of protein expression from fractionated primary cortical neurons treated with A�o (100 nM) and then for 15 min with Bic/4-AP.Presynaptic protein (synaptophysin), postsynaptic proteins (PSD-95, GluA1,GluN2A, GluN2B,Fyn), cytoskeleton protein (actin), and tau in PSD-fraction (Triton-insoluble fraction) were analyzed. C,Quantitative analysis of PSD-95, GluA1, GluN2B, GluA1, Fyn, actin, and tau in PSD fraction (mean� SEM, 2-tailed Student’s t test, N � 7). D, Confocal imaging of cortical neurons (14 DIV) transfectedwith GFP membrane. Top, Untreated neurons. Scale bar, 10 �m. Middle and Right, higher magnification of GFP membrane in dendrites (scale bar, 5 �m) and dendritic spine (designated box in themiddle) from a neuron showing the effect of 15 min of A� treatment on spines followed by 15 min of Bic/4-AP. Scale bar, 1 �m.

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modulate tau-dependent neurodegeneration in the Thy-Tau22Drosophila model (Blard et al., 2007). However, these studiesprovide no direct evidence for a physiological interaction be-tween tau and myosin. Last, tau is unlikely to enter spinesthrough simple diffusion because very little tau is present inspines before synaptic activation or exposure to A�. Therefore,we favor a combined mechanism that involves tau diffusion alongthe microtubule lattice, as demonstrated by Hinrichs et al.(2012). When microtubules are briefly entering the dendriticspines during activation, tau diffusion guided by the microtubulelattice may promote spine distribution independently of directedmotor-dependent transport. The importance of tau transloca-tion to spines warrants further investigation of the many poten-tial mechanisms, which may be different depending on theevoking stimuli.

Once located in the spines, tau may contribute to spine mor-phology. Such a role is supported by previous observations thatthe silencing of tau expression using small hairpin RNA producedspine loss in cultured neurons (Chen et al., 2012).

The stimuli that led to tau translocation into spine heads alsoinduced the long-lasting modifications of the postsynaptic ele-ment that characterize LTP. The tau increase in the PSD-enrichedfraction is concomitant with enrichment in PSD95 and GLUA1,both of which are necessary characteristics of synaptic potentia-tion (Ehlers, 2003; Steiner et al., 2008; Kessels and Malinow,2009). Following the same behavior of well known plasticity-related synaptic proteins such as GluA1, PSD-95, or actin sug-gests that tau may contribute to the functional modifications thatsustain the enhanced synaptic strength associated with LTP. Therole of tau in synaptic plasticity could be due to its ability totransport the Src kinase Fyn to synaptic targets (e.g., GluN2Bsubunits of the NMDA receptor). In accordance with this hy-pothesis, tau KO mice or those expressing tau without MT-binding domains display no Fyn in the PSD fraction (Ittner et al.,2010). The importance of Fyn in synaptic plasticity processes isunderscored by the decreased LTP observed in Fyn KO mice(Grant et al., 1992; Kojima et al., 1997).

Tau interacts with F-actin in both purified protein assay andspines. This interaction was identified and characterized for thefirst time in similar purified protein assay involving tau MT-binding domains and the portion of the proline-rich domain thatinteracts directly with F-actin (He et al., 2009). Similarly, thisinteraction has been identified in PC12 cells (Yu and Rasenick,

2006) and in a Drosophila model of AD (Fulga et al., 2006). Theconsequences of the interaction between tau and F-actin are astabilization of actin filaments and their organization into bun-dles. Here, we show that interaction between F-actin and tau isincreased during synaptic activation. As revealed by LifeAct flu-orescence, filamentous actin displays a higher concentration inthe spine heads. The potential interaction of tau and filamentousactin in the shaft and then the translocation to the synapse ispossible, although we favor a molecular mechanism that involvesinteraction of tau/actin in the spine because of the presence of thisdense spine actin cytoskeleton. The role of actin in the structuralreorganization necessary for synaptic plasticity has been studiedextensively (for review, see Hotulainen and Hoogenraad, 2010).Indeed, modulation of actin dynamics toward F-actin complexstructures can be considered as one of the key elements respon-sible for the activity-dependent modification of spine morphol-ogy during synaptic plasticity (Star et al., 2002). Therefore, taumay be important for synaptic plasticity because of its ability toinfluence F-actin structure or stability. F-actin as causal to tautranslocation is central to our hypothesis. The fact that jas-plakinolide treatment induces a translocation of tau to spineheads strongly supports a causal relation between these events(i.e., direct stabilization of F-actin produces tau translocation).

Finally, when we reproduced pathological conditions ob-served in AD by exposing cortical neurons to A�o, we observed atranslocation of tau to the synapse despite the lack of synapticactivation. This result is consistent with abnormal tau accumula-tion in dendritic spines (Hoover et al., 2010). Recently, similarsynaptic accumulation of tau has been described in AD patients,further strengthening the hypothesis that abnormal synaptic lo-calization of tau may be involved in the synaptic dysfunctionobserved in AD (Roberson et al., 2011; Tai et al., 2012). UnderA�o exposure, tau still interacts with F-actin once in the postsyn-aptic compartment. Our FRAP data highlighted a more dynamicpool of synaptic tau under A�o compared with that evoked bysynaptic activation, which suggests that tau interactions with itspotential partners in the synapse, such as F-actin, are disturbed.This likely disrupts tau-related modification of the postsynapticcontent observed during synaptic plasticity. Therefore, synapticactivation in the presence of A�o did not lead to increased levelsPSD95 or GluA1 in the PSD fraction and precluded the establish-ment of long-lasting modification of synaptic strength, which is

Figure 8. A�o treatment on primary cortical neuron cultures increases the interaction of F-actin with tau, which is reduced after synaptic activation. A, Representative Western blot showing theeffect of 30 min A�o (100 nM) and the effect of 15 min pretreatment with A�o (100 nM) then 15 min of Bic/4-AP treatment on tau associated with F-actin in cultured cortical neurons. F-actin wascollected after treatment and precipitated with phalloidin. B, Western blot analysis of actin and tau in control, A�o (100 nM), and A�o (100 nM) Bic/4-AP 15 min condition (**,*p � 0.05 vscontrol, 1-way ANOVA; #p � 0.05 vs A�o, N � 5 independent cultures).

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consistent with the exhaustive reports of A�o impairing LTP(Shankar et al., 2007; Selkoe, 2008; Li et al., 2011).

The difference in tau behavior depending on the stimulusdriving it to the synapse is puzzling and may rely on the variationin the phosphorylation profile (Allyson et al., 2010; Mondragon-Rodríguez et al., 2012). It is now well accepted that tau functionand localization to the membrane, nucleus, synapse, or axons ismodulated by phosphorylation (Mandell and Banker, 1996; Sul-tan et al., 2011; Pooler et al., 2012; Hoover et al., 2010). Weshowed that A�o and synaptic activation display differences inthe phosphorylation of two tau phospho-epitopes. Under synap-

tic activation, PSD-located tau is phosphorylated on threonine205, whereas it is largely dephosphorylated under A�o exposure.In contrast, serine 404 is not differentially phosphorylated be-tween these two experimental conditions. The nonphosphoryla-tion incompetent mutated tau exhibited drastic differentfeatures, with wild-type tau strengthening the importance ofthese phosphorylations in tau behavior.

In summary, we provide evidence that, under physiologicalstimulations, tau may be involved in the molecular cascade lead-ing to synaptic plasticity. A�o renders the synapse unable to es-tablish plasticity-related modifications of the postsynaptic

Figure 9. The dynamics of tau in spines changes is distinct between translocation inducing synaptic activation from A� exposure and could be phosphorylation dependent. A, Timelapse of a spine pretreated with Bic/4-AP containing EGFP-tau and showing FRAP. B, Representative FRAP curves (%FRAP normalized to the prebleached intensity of the spine ofEGFP-Tau-transfected neurons) of Tau WT (EGFP-Tau 2N4R) after 45 min of Bic/4AP (green, n � 21) or 45 min after A�o application (red, n � 24) and TauT205A after 45 min of Bic/4AP(blue, n � 21). C, Percentage of EGFP-tau fluorescence or EGFP-tauT205A that was stable (white), dynamic (gray), or unbleached (black). Statistical analysis was performed by 2-tailedt test, ***p � 0.0001. D, Representative Western blot of Tau phospho-Threonine 205 (p-Thr205), phospho-Serine 404 (p-Ser 404), and tau total from PSD-enriched fractions for control,Bic/4-AP (30 min), A� (30 min), and A� (15 min)Bic/4-AP (15 min) conditions. E, Quantifications of p-Thr 205 and p-Ser 404 under control, Bic/4-AP, A�, or A� Bic/4-AP conditions(mean � SEM, **,*relative to control; # # #relative to Bic/4-AP, �relative to A�, p � 0.05, 1-way ANOVA with Bonferroni’s posttest, N � 5 independent cultures). F, Confocal imagingof cortical neurons (14 DIV) cotransfected with EGFP-Tau S404A and LifeAct-RFP. Left, Spatial repartition of EGFP-TauS404A (green) and LifeAct-RFP (red) in untreated neurons. Scale bar,5 �m. Right, Higher magnification of EGFP-Tau S404A and LifeAct-RFP in dendritic spine neuron after A� treatment (15 and 30 min, designated box in the left) by 15 and 30 min of A�o100 nM treatment. The merged image shows no colocalization of F-actin and tau. Scale bar, 1 �m. G, Quantification of the fluorescence intensity of EGFP-TauS404A in the head of spinesduring the A�o treatment. The graph represents the evolution of �F/F0 generated by EGFP-Tau under control conditions (black), EGFP-Tau (red), and EGFP-TauS404A (pink) induced byA�o 100 nM treatment. Statistical analysis was performed by 2-way ANOVA followed by Bonferroni’s posttest (mean � SEM, ***p � 0.001, control n � 40, N � 3 independent cultures;Tau-WT A�o 100 nM n � 79, N � 5; Tau S404A n � 98, N � 3 independent cultures).

Frandemiche et al. • Activity-Dependent Tau Protein Translocation J. Neurosci., April 23, 2014 • 34(17):6084 – 6097 • 6095

content. The disruption of the activity-driven relocalization oftau to the synapse by A�o could be one of the synaptotoxic stepsin the progression of AD. The mechanism responsible for thealtered localization of tau may represent a novel therapeutic ap-proach for tauopathies, including AD.

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